Nanoparticle and microparticle based detection of cellular products

ABSTRACT

Embodiments of the present invention relate to devices and methods for detecting cellular products using detection particles having product-specific detection reagents and having a characteristic spectral feature. In particular, devices and methods are provided for measuring secreted cellular products including cytokines. Detection substrates, include microwells having product-specific capture reagents thereon or comprising hydrophobic membranes are described having greater capability to detect products from individual cells in a mixture of heterogeneous cells. With the use of multiple detection particles, multiple cellular products can be detected in a single well. Additionally, using the inherent spectral properties of detection particles, no enzymatic reactions are needed to visualize a secreted product, thereby increasing the sensitivity, reproducibility and ease of use.

RELATED APPLICATION

This application claims priority under 35 U.S.C. section 119(e) to U.S.Provisional Patent Application Ser. No. 60/489,451, filed Jul. 23, 2003,Paul Lehmann and Alexey Y. Karulin, titled “Microsphere Based Detectionof Cellular Products.” This application is herein incorporated fully byreference.

FIELD OF THE INVENTION

The present invention relates to devices and methods for detectingsecreted cellular products, and in particular for measuring secretedsoluble cell products using nanoparticles and/or microparticles withassociated capture reagents that recognize the cellular product.

BACKGROUND

Over the last thirty years, the immune system has been studied withbetter and better laboratory tools. Still, most knowledge of the immuneresponse concerns antibody formation. This is understandable given thatantibodies, including specific antibodies, are easily detectable inquantity in the serum of immunized individuals. Antibodies are productsof B-lymphocytes (“B cells” or “B-cells”). Antibody production byindividual B cells (as well as cells fused with B cells such ashybridomas) is also readily achieved in vitro using a variety of tests,including the ELISA spot assay (also called “ELISPOT” for “enzyme-linkedimmunospot”). See Segwick, J. D. and Holt, P. G., “A solid-PhaseImmunoenzymatic Technique for the Enumeration of SpecificAntibody-Secreting Cells,” J. Immunol. Methods 57:301-309 (1983). Seealso Mazer, B. D. et al., “An ELISA Spot Assay for Quantitation of HumanImmunoglobulin Secreting Cells,” J. Allergy Clin. Immunol. 88:235-243(1991).

In a conventional B cell ELISA spot assay, standard,commercially-available plates are coated overnight with antigen oranimal antibody. In the case where antibody is used, it is typically an“anti-antibody” (e.g., goat antibody reactive with human IgG, IgE, IgM,etc.). After blocking overnight, B cells are introduced in the wells.Following a sufficient culture period, the wells are washed free of thecells and an antibody-enzyme conjugate is added. The plates are thendeveloped using substrate for the enzyme of the conjugate. Spots arecounted using a microscope. The lowest amount of detectable antibody istypically in the range of 10 to 50 picograms. See e.g., Renz, H. et al.,“Enhancement of IgE Production by Anti-CD40 Antibody in AtopicDermatitis,” J. Allergy Clin. Immunol. 93:658-668 (1994).

In contrast to the antibody response, the response of T-lymphocytes(“T-cells” or T Cells”) to antigen (including the antigen of pathogens)can not be easily monitored due to the fact that antigen reactive Tcells occur in low frequencies and the fact that their secretoryproducts are not typically stable (i.e. have a short half-life). Indeed,even in hyperimmunized individuals, antigen reactive T cells constituteI in 10,000 cells or less in the peripheral T cell pool, for example,the T cells in circulating blood. Thus, T cells usually act beyond thedetection limits of conventional assay systems (such as proliferationassays).

As a consequence of this, there are few sensitive, reliable and rapidtechniques at present available that would reliably measure whether apatient has generated a T cell response to a particular pathogen, suchas HIV. There is no reliable assay that can detect whether a T cellresponse to HIV proteins has been generated, what proteins of the virusare primarily targeted, and which determinants within that protein areimmunodominant. There is also no reliable method available for testingthe magnitude of the anti-viral T cell response (clonal sizes) and itsquality (e.g. whether the response is pro- or anti-inflammatory).

Heterogeneity of T cells, their products and the mode of functionprovide great challenges (particularly as compared to B cells). Withrespect to mode of function, T cells can act in different subpopulationsthat utilize strikingly different effector functions. T cell responsescan be pro-inflammatory T helper 1 type, Th1, characterized by thesecretion of interferon gamma (IFNγ) and interleukin 2 (IL-2). Th1 cellsare critical for the cellular defense and provide little help forantibody secretion. (Strong Th1 responses are usually associated withpoor antibody production, which highlights the importance of directlymeasuring the T cell response instead of relying on antibodymeasurements.) The other class of T cell responses is antiinflammatory,mediated by Th2 cells that produce IL-4, 5, 10, but no IL-2 or IFNγ. Th2cells are the helper cells for antibody production. CD4+ and CD8+ cellsboth occur in these subpopulations: Th1/Th2:CD4, TC1/TC2:CD8.

Importantly, for each type of infection there is an “appropriate” (anddifferent) type of T cell response (e.g., Th1 vs. Th2, CD4+ vs. CD8+)that clears the infectious agent but does not cause excessive tissuedestruction to the host. It is detrimental to the host if an“inappropriate” type of T cell response is engaged (Th1 instead of Th2or vice versa). Thus, there is a strong need for assessing the host's Tcell immunity to the virus to understand the host-virus interplay and todesign vaccines. An ideal assay should permit monitoring all of thecritical features of the T cell response: first, the existence of aresponse, i.e., that effector cells have been generated, second thenature of the effector cells as Th1 or Th2 type cells, and finally themagnitude of the response.

Some attempts have been made to apply the B-cell ELISA spot technologyto T cells. However, the conventional cytokine ELISA spot assay has notbeen a more sensitive tool than alternative assays (e.g. proliferationassays), displaying high background and generally a weak signal. Theconventional ELISA spot assay for 1 T cells involves plates containingnitrocellulose membranes that are pre-coated with a capture antibodyspecific for the cytokine to be detected. See e.g., Taguchi, T. et al.,“Detection of Individual Mouse Splenic T Cells Producing IFNγ and IL-5Using the Enzyme-Linked Immunospot (ELISPOT) Assay,” J. Immunol. Methods128:65-73 (1990). See also Fujihashi, F. et al., “Cytokine-SpecificELISPOT Assay,” J. Immunol. Methods 160:181-189 (1993). See alsoMiyahira, Y. et al., “Quantification of Antigen Specific CD8+ T CellsUsing an ELISPOT Assay,” J. Immunol. Methods 181:45-54 (1995). T cellsare plated with the test antigen and start to secrete the type ofcytokine they are programmed to produce. As the cytokine is released, itis captured around the secreting cells by the plate bound antibody.After 24 h the cell culture is terminated, cells are removed and theplate-bound cytokine is visualized by a second antibody and an enzymaticcolor reaction.

Ideally, each product-producing cell will be represented as an ELISAspot. However, with conventional assays, sensitivity does not exceedcytokine measurements in the supernatant by ELISA (cytokine measurementsin culture supernatants provide a positive result only if more than 1000cells are present per well). The quantification of the data is alsoproblematic because of background problems and the subjective, visualevaluation. Moreover, because enzymatic reactions are sensitive to time,reagent quality and reaction conditions, there can be variations inresults obtained using conventional ELISA or ELISPOT methods. Moreover,conventional 2 or 3-color systems used with conventional ELISA orELISPOT methods cannot separately resolve individual spectral featuresof each colored detection reagent. In conventional multicolor assays,development of colors typically is made sequentially, one after another,each followed by washing steps. This procedure is time consuming andbecause of its complexity is difficult to carry out. Additionally,substrates are not typically fully transparent, so a second substratemay mask an underlying substrate. Moreover, as more substrates are used,the background becomes darker and less controlled. The above problemswith conventional ELISA methods therefore make assays of multiplecellular products difficult or impossible.

There is a great need for better assays to measure secreted soluble cellproducts. Specifically, there is a need for devices and methods withgreater capability to detect cytokines from individual cells in amixture of heterogeneous cells, and for methods that can detect multipledifferent secreted cytokine cell products within mixtures of suchproducts, and for methods that have increased reliability andreproducibility.

SUMMARY OF THE INVENTION

The present invention relates to devices and methods for detectingcellular products, and in particular for measuring secreted cellproducts using nanoparticles, microparticles or combinations ofnanoparticles and microparticles (collectively termed herein “detectionparticles”) that are associated with capture reagents raised against thecellular product (also know as an “analyte”) of interest.

In general, embodiments of this invention comprise a surface having acapture reagent thereon that is specific or selective for the analyte ofinterest. When an analyte binds to the capture reagent, a detectionreagent is then used to bind to the analyte/capture reagent complex. Thedetection reagent can bind either to the analyte (on a different portionof the molecule than that which the capture reagent binds) or to thecomplex itself (e.g., antibodies raised against a capturereagent/analyte pair). Such types of assays are referred to herein as“direct” assays. In some embodiments, the detection reagent includes ananoparticle or microparticle that has a uniquely defining feature thatpermits identification and/or quantitation of the amount of thedetection reagent present. The identifying feature can be a fluorescentmarker, an absorption marker or a phosphorescent marker.

In alternative embodiments, a secondary binding reagent can be used tobind to the capture reagent/analyte pair. In these embodiments, thesecondary binding reagent can also have a moiety that is recognized by adetection reagent associated with a nanoparticle or microparticle. Suchtypes of assays are “sandwich” or “indirect” assays.

Regardless of whether the detection is “direct” or “indirect,” once adetection reagent is associated (or bound) to the analyte, microscopicor spectroscopic analysis of the detection reagent can be made.

In some embodiments, the surface is a plastic or glass surface of a cellculture dish. In other embodiments, the surface can be a membrane,either a hydrophilic or hydrophobic membrane.

Because the markers used as part of this invention have clearly separatespectral features, one can detect multiple secreted soluble cellproducts simultaneously, thereby permitting increased speed of analysisand providing ability to compare differences between secreted productsfrom the same cell culture. Further, because no enzyme reaction isneeded, the assay devices and methods of this invention have improvedreliability and accuracy.

DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows one embodiment of the method of the presentinvention. FIG. 1A is a schematic diagram showing a capture reagent(“Y”) bound to a surface within a test well. FIG. 1B is a schematicdrawing showing cells in solution secreting soluble products (solidblack diamonds) binding to the immobilized capture reagent. FIG. 1C is aschematic diagram showing surface bound secreted product. FIG. 1D is aschematic diagram showing binding of four detection reagents coupleddirectly to 4 different types of detection particles. Each detectionparticle has a characteristic spectral feature. FIG. 1E is a schematicdrawing showing binding of four detection reagents coupled indirectly toeach of the 4 detection particles via a secondary detection reagent.

FIG. 1F depicts an alternative embodiment 101 having two types ofcapture reagents 108 and 109 that are specific for different secretedcellular products. FIG. 1G depicts an embodiment wherein two cells 116and 113 are stimulated to produce products 120 and 122, respectively.FIG. 1H depicts an embodiment of the invention in which cells 116 and113 have been removed, leaving their secreted products 120 and 122 boundto capture reagents 108 and 109, respectively. FIG. 1I depicts multipleproduct assay embodiment in which detection particles 132 and 138 areshown bound to products 120 and 122, respectively.

FIG. 2 depicts a schematic drawing of an embodiment of this invention inwhich a number of different wells are decorated with detectionreagent-bound particles of this invention.

FIG. 3 depicts an assay according to an embodiment of this invention inwhich two types of detection particles are used.

FIG. 4 depicts a diagram for producing purifiedstreptavidin/biotinylated detection particles according to an embodimentof this invention.

DETAILED DESCRIPTION

The present invention relates to devices and methods for detectingcellular products, and in particular for measuring secreted/releasedcellular products, including cytokines. In general, the devices andmethods of this invention can be used to detect any analyte for whichthere is a capture reagent to associate the cellular product with adetection substrate, a detection reagent that can be attached to amicroparticle or nanoparticle (herein termed a “detection particle”). Todetect an analyte, the analyte to be detected is associated with adetection substrate, such as a microwell of a plate. In someembodiments, the detection substrate includes a hydrophobic membrane orsurface to which a hydrophobic analyte may adhere due to physicalinteraction with the substrate. In other embodiments, a detectionsubstrate can include one or several analyte-specific ligands, such asantibodies, analyte receptors or combinations of these. Suchanalyte-specific capture reagent can be associated (e.g., throughchemical linking to the surface). Once the analyte is associated withthe capture reagent or directly binds to detection surface, a detectionparticle having a characteristic detection reagent (e.g., antibodyanalyte specific receptor) is conveniently used. In some embodiments,the detection reagent is an antibody, such as a monoclonal antibody or apolyclonal antiserum. In additional embodiments, a capture reagent is aligand specific for the analyte of interest. In still other embodiments,a detection particle includes a moiety that can be spectroscopicallyidentified, such as by color, fluorescence, or phosphoresence or otheroptical feature associated with the detection particle. In someembodiments, the characteristic spectrographic feature is a moiety thatis added to the detection particle after its manufacture. In otherembodiments, the spectrographic feature is inherently part of thedetection particle.

Detection Substrates

The type of detection substrate is not crucial. In some embodimentshydrophilic surfaces can be used. Hydrophilic surfaces can include cellculture dishes of plastic or glass, or hydrophilic membranes (e.g.,cellulose). In these embodiments, the analyte specific capture reagentcan be attached to the detection surface using chemical means. Means forattaching proteins to a surface are well known in the art (for exampleusing streptavidin/biotin) and need not be described herein further. Insome embodiments, regular tissue culture plates can be treated for highprotein absorption and/or increased surface area (e.g., corrosion).

The present invention also includes detection substrates that arehydrophobic. In some embodiments a membrane having some hydrophobiccharacteristics can be used. These embodiments can be especially usefulfor detection of hydrophobic analytes that need no analyte-specificcapture reagent to become adhered to the detection substrate. In oneembodiment, membranes are weakly hydrophilic and display advancingcontact angles for water between approximately seventy (70) andapproximately ninety (90) degrees. In another embodiment, the membranesare hydrophobic and display advancing contact angles for water betweenapproximately ninety (90) and approximately one hundred and thirty (130)degrees. In another embodiment, the membranes are very hydrophobic anddisplay advancing contact angles for water greater than approximatelyone hundred and thirty (130) degrees.

Capture Reagents

In other embodiments of the invention, a detection substrate can be amicrowell pre-coated with a capture reagent selective for a particularcytokine (see FIG. 1A). It is not intended that the present invention belimited by the nature of the capture reagent. In certain embodiments, acapture reagent is a binding ligand such as a capture antibody specificfor the materials (analytes) to be detected. In certain embodiments, twoor more different analytes can be detected simultaneously (e.g.,anti-IFNγ, mAb1 or anti-IL5 mAb or with both simultaneously for a twocolor assay). In other embodiments, multiple analytes can be detectedsimultaneously using a single assay substrate. Analyte receptormolecules, such as recombinant receptors, can also be used as capturereagents. Additionally, capture of an analyte can be accomplished usingphysical association, for example, hydrophobic interaction of non-polaror amphipathic analytes on surfaces that are hydrophobic. Thus, all ofthe capture mechanisms are herein termed “capture means.”

Detection Particles

Once a secreted cellular product is associated with a capture reagent ora membrane, one or more detection particles can be added. As analternative, molecules released by dying or injured cells, such as LDH,could be measured using similar methods. A detection particle can becomeassociated with the analyte, a capture reagent bound to the analyte, orwith an analyte/capture reagent complex. Thus, an assay for thesecretion of a cellular product includes detection of a type ofdetection particle that is associated with that particular secretoryproduct. For example, Quantum Dot Corporation produces particles thatcan be adapted for use with the devices and methods of this invention.Further description of such particles is provided in Wu et al., NatureBiotechnology 21(4):41-46 (January 2003) and Watson et al.,Biotechniques 34:296-303 (February 2003), each reference incorporatedherein fully by reference.

The only requirement for a detection particle material is that it besuitable for associating a capture reagent with the detection particlewithout loss of the capture reagent's ability to bind to an analyte, andthat it be compatible with the cells or other components of the testsystem. Thus, detection particles can comprise organic polymers,inorganic semiconductors or heavy metals. For example, organic polymersinclude polycarbonates, polyacrylamides, polyacroleins, polystyrenes andthe like. Inorganic semiconductors include silicon dioxide-containingmaterials, cadmium selenide (CdSe) and the like. The only requirementfor polymers and semiconductors is that they have a characteristicemission or absorption spectral feature that permits their detection.Thus, colored organic polymers can appear blue, green or red, or inbetween. In certain embodiments the emission “color” can depend upon thesize of the detection particle. Thus, very small particles can fluorescein the ultraviolet (UV) range, particles of intermediate size canfluoresce in the green portion of the spectrum, and large detectionparticles can fluoresce in the red or infrared (IR) portions of thespectrum.

The size or shape of a detection particle is not, in general limited.However, it can be convenient to use particles of sufficient size andshape to produce a detectable signal that defines the particle. Thus,nanoparticles, microparticles, nanospheres, microspheres or other shapedparticles can be used. As used herein, the term “nanoparticle” generallyrefers to particles having sizes with a lower limit of about 90 {acuteover (Å)} and an upper limit of about 500 {acute over (Å)}. The term“microparticles” generally refers to particles having sizes in the rangeof about 0.05μ to about 5μ.

Heavy metals include gold, silver, platinum, palladium, lead and thelike. It can be appreciated that any heavy metal that can providecontrast between the unlabeled portion of the detection substrate andthe detection particle can be advantageously used.

Thus, to associate metal detection particles, sulfhydryl reagents, suchas thiols, and in some embodiments, di-thiols can be used. It is knownthat certain metals, such as silver, gold and the like, can bind tosulfhydryl (—SH) groups. It is also known that certain proteins (e.g.,antibodies and receptor proteins) can bind to —SH groups. Thus, by theuse of a di-sulfhydryl reagent such as a di-thiol, detection reagentscan be associated with detection particles, thereby forming a detectionreagent/capture reagent detection particle. However, it can beappreciated that other types of attachment can be used.

It is also known that certain plastics, such as styrenes, polycarbonatesand the like can bind to proteins, such as antibodies and receptors.Regardless of the type of detection particle used or the type ofcoupling chemistry used to associate the detection particles with thecapture antibody, this invention contemplates using all types ofmaterials and coupling chemistries.

Complexes of Detection Reagents and Detection Particles

In certain embodiments, a detection reagent can be coupled convenientlyto a detection particle. In some of these embodiments, streptavidin andbiotin can be used. In general, a detection particle having straptavidinthereon is reacted with a biotinylated detection reagent (e.g., anantibody) to produce a complex suitable for use in an assay of thisinvention. One advantage of streptavidin biotin systems is that one canvary the detection particle and the detection reagent as desired withthe same coupling chemistry. It is therefore easy to produce a widevariety of detection particle/detection reagent complexes to suit anyparticular need.

In certain embodiments, such complexes can be made and purifiedconveniently using a novel purification method. An illustrative exampleof the method is presented herein as Example 8.

Assays Using Detection Particles

To perform multiple analyses using a single sample, different types ofdetection particles can be associated with different analytes or capturereagents. Each type of detection particle can be independently detectedusing a characteristic spectral feature. By detecting the association ofa particular type of detection particle with a membrane, multiplecellular products can be simultaneously detected. FIG. 1 depicts anembodiment of the invention.

In some of the descriptions that follow, features of the devices andmethods of this invention are described with reference to T-cellcytokines. It is not intended that the scope of this invention belimited to detection of cytokines. Rather, the full scope of theinvention includes products secreted by other cell types, for exampleonly, B-cells, epithelial cells, muscle cells, nerve cells, bacterial,fungi, stem cells, endothelial cells, plant cells and the like.

In certain embodiments, freshly isolated, primary cell populations(e.g., lymph node, spleen cells, etc.) are subsequently plated (see FIG.1B) with the test antigen, (e.g., HIV protein or peptide) and controlcultures contain irrelevant antigens or peptides. Since the primary cellsuspensions contain abundant antigen-presenting cells (APCs) to processand present the antigen, specific T cells become activated and start tosecrete the type of cytokine they are programmed to produce. As thecytokine is released, it is captured around the secreting cells by theplate-bound capture reagent (see FIG. 1B). After a suitable cultureperiod (e.g., between approximately 30 minutes and 48 hours), the cellculture is terminated and the cells are removed (e.g. by washing),leaving the captured, plate-bound secreted product (see FIG. 1C).

It is not intended that the present invention be limited by the natureof the detection reagent. In one embodiment, the detection reagent is asecond cytokine-binding ligand (e.g., antibody) free in solution that isconjugated to a detection particle having a secondary detection reagent(e.g., secondary antibody that binds the first detection reagent). Inother embodiments, the present invention contemplates the use ofdirectly labeled detection reagents (e.g. antibodies coupled todetection particles). Each cytokine-producing cell will be representedas a spot. In broad aspects of this invention, detection by detectionparticles may appear similar to an ELISA spot (“ELISPOT”). However, withthe use of detection particles, it is not necessary to use an enzymesubstrate, as needed for an ELISA assay.

In still other embodiments, the detection particle can be directlylabeled with a fluorochrome (e.g., FITC, PE or texas red) or withcolored beads (different colors for different secretory products). Itcan also be labeled with a ligand such as streptavidin-biotin, which canbe detected with tertiary reagent that is labeled as above (i.e. with afluorochrome or bead).

While certain embodiments of the present invention employ, as a firstcapture reagent, cytokine-binding antibodies bound to a detectionsurface, the present invention also contemplates the binding of solubleproducts directly to a membrane without the use of capture reagents. Instill other embodiments, the present invention contemplates non-antibodyligands such as cytokine receptors as a capture ligand.

The present invention also contemplates testing devices comprising aplurality of areas on a detection substrate, such as a culture dishincluding 96-well plates or 360 microwell plates,

In a specific embodiment, the present invention contemplates a method ofdetecting secreted/released T cell cytokines or other products of acell, comprising: a) providing: i) a detection substrate or detectionsurface comprising at least one capture reagent; ii) a cell populationcapable of secreting products or inducing their release, b) adding thecell population to a microwell under conditions such that said cellsecretes/releases molecules that bind to the capture reagent; c) addinga detection particle, which binds to the cellular secreted product, and(d) detecting the presence of the detection particle.

The present invention further contemplates embodiments wherein saiddetection substrate further comprises, prior to said adding of step (b),a test antigen (such as a peptide). Such antigens can be used to testeither the specificity of reaction of the analyte to the capture reagentor the specificity of reaction of the detection reagent to the analyte,capture reagent or the analyte/capture reagent complex.

Detection of Cell Products, Including Cytokines

With the present invention one can obtain very clear signals (which arereadily suited for image analysis) where previously signals wereambiguous or not detectable at all. One can detect simultaneouslyseveral cytokines being produced by T cells. Indeed, the detectingseveral secretory T cell products at single cell resolution represents amajor development in monitoring T cell responses.

While particularly suitable for measuring T cell products such ascytokines, it is also not intended that the invention be limited to thetype of sample. The present invention can be employed with success withall types of liquid samples, including various different suspensions ofbiological material.

In accordance with one embodiment 100 of the invention, as depicted inFIG. 1A a microwell 104 is pre-coated with a first capture reagent 108.In some embodiments, the capture reagent is a cytokine capture antibodyspecific for the cytokine to be detected (e.g., anti-IFNγ mAb oranti-IL5 mAb or with both simultaneously for the two color assay) orreceptors. As depicted in FIG. 1B, certain antigen-exposed cells 112 donot secrete any soluble cell product. However, as shown in FIG. 1C,other, antigen-stimulated cells 120 produce soluble cellular product 124into the medium. As the product 124 is released, some is captured aroundthe secreting cells by the capture reagent 108. After a suitable cultureperiod (e.g., between approximately 30 minutes and 48 hours), the cellsare removed (e.g. by washing), leaving the captured, plate-boundsecreted product (see FIG. 1C). FIG. 1D depicts an embodiment of thisinvention in which four different detection particles are shown (128,132, 136, and 140) respectively. Each of detection particles 128, 132,136, and 140 are associated with a detection reagent 124. In thisFigure, detection particles 128, 132, 136, and 140 each becomeassociated with the same analyte 120. The differences in the detectionparticles 128, 132, 136, and 140 reflect differences in characteristicspectrographic features, thereby permitting their differentialdetection. It can be appreciated that there is no stringent upper limitto the numbers of different cell products (analytes) that can bedetected using methods of this invention. In some embodiments, it can bedesirable to detect only a single cell product. In other embodiments, itcan be desirable to detect 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, such as15, 20, 30, 50 or even 100 different products in a single assay. Theonly requirement is that sufficient cell product be present in thesample to permit sufficiently intense spectrographic signals to beproduced by each of the multiplicity of detection particles.

FIG. 1E depicts an alternative embodiment of this invention in which 4detection particles are shown associated with the analyte via asecondary detection reagent 125 (e.g., a secondary antibody).

FIG. 1F depicts an alternative embodiment 101 having two types ofcapture reagents 108 and 109 that are specific for different secretedcellular products.

FIG. 1G depicts an embodiment wherein two cells 116 and 113 arestimulated to produce products 120 and 122, respectively.

FIG. 1H depicts an embodiment of the invention in which cells 116 and113 have been removed, leaving secreted products 120 and 122 bound tocapture reagents 108 and 109, respectively.

FIG. 1I depicts a multiple product assay embodiment in which detectionparticles 132 and 138 are shown bound to products 120 and 122,respectively.

FIG. 2 depicts an embodiment 200, which represents a portion of amultiwell plate having 6 columns (1 to 6) of wells in 3 rows (A to C).Wells A1-A6 have no labeled materials therein. Well B3 is depicted ashaving detection particle 236 therein, and well B4 is depicted as havingdetection particle 228 therein. Well C3 is depicted as having detectionparticle 240 therein, and well C6 is shown as a positive control.

TABLE 1 Name Abbr. Type Specific Types Interferons IFN alpha LeukocyteInterferon beta Fibroblast Interferon gamma Macrophage Activation FactorInterleukins IL-1 1 alpha Endogenous Pyrogen 1 betaLymphocyte-Activating Factor 1 ra IL-1 Receptor Antagonist IL-2 T-cellGrowth Factor IL-3 Mast Cell Growth Factor IL-4 B-cell Growth FactorIL-5 Eosinophil Differentiation Factor IL-6 Hybridoma Growth Factor IL-7Lymphopoietin IL-8 Granulocyte Chemotactic Protein IL-9 MegakaryoblastGrowth Factor IL-10 Cytokine Synthesis Inhibitor Factor IL-11 StromalCell-Derived Cytokine IL-12 Natural Killer Cell Stimulatory Factor TumorTNF alpha Cachectin Necrosis beta Lymphotoxin Factors Colony CSF GM-CSFGranulocyte-macrophage Stimulating Colony-Stimulating Factor FactorsMp-CSF Macrophage Growth Factor G-CSF Granulocyte Colony- stimulatingFactor EPO Erythropoietin Transforming TGF beta 1 Cartilage-inducingFactor Growth Factor beta 2 Epstein-Barr Virus-inducing Factor beta 3Tissue-derived Growth Factor Other Growth LIF Leukemia Inhibitory FactorFactors MIF Macrophage Migration- inhibiting Factor MCP MonocyteChemoattractant Protein EGF Epidermal Growth Factor PDGFPlatelet-derived Growth Factor FGF alpha Acidic Fibroblast Growth Factorbeta Basic Fibroblast Growth Factor ILGF Insulin-like Growth Factor NGFNerve Growth Factor BCGF B-cell Growth Factor

In addition to those products listed above in Table 1, other productsinclude IL-13, IL-15, IL-16, IL-17, IL-18, IL-23, GranzymeB, Perforinand RANTES.

A captured molecule can be visualized by a detection reagent associatedwith a detection particle. It is not intended that the present inventionbe limited by the nature of the detection reagent or the detectionparticle. In one series of embodiments, the detection reagent is asecond cytokine binding ligand (e.g., antibody or receptor) free insolution that is conjugated to a detection particle. Regardless of thetype of detection reagent used, once the cell product is associated witha specific type of detection reagent and detection particle, detectionof the detection particle reveals the presence of the analyte.

It is not intended that the present invention be limited by the natureof the product to be detected. Cytokines are hormone-like substancessecreted by a wide variety of cells, including (but not limited to)lymphocytes (e.g., T cells), macrophages, fibroblasts, and endothelialcells. It is now known that cytokines consist of a broad class ofglycoproteins that have the ability to regulate intercellularcommunication (e.g., cell-cell interaction) in both normal andpathologic situations. Cytokines generally contain from approximately 60to 200 amino acid residues, with a relative molecular weight of between15 and 25 kd. At least 35 distinct cytokines have been elucidated (seeTable 1 above).

There is also a family of chemo-attractant cytokines known as“chemokines.” See e.g. T. J. Schall and K. B. Bacon, “Chemokines,leukocyte trafficking, and inflammation” Curr. Op. Immun. 6:865-873(1994). These molecules share structural similarities, including fourconserved cysteine residues which form disulfide bonds in the tertiarystructures of the proteins. The present invention contemplates employingthe devices and methods of the present invention for detecting secretedchemokines.

Role of many cytokines are now known. For example, interleukin-1α andinterleukin-1β (IL-1α and IL-1β) are distantly related polypeptidehormones that play a central role in the regulation of immune andinflammatory responses. See D. P. Cerretti et al., U.S. Pat. Nos.4,894,333 and 4,879,374, each hereby incorporated by reference. Thesetwo proteins were originally both classified as IL-1, based on a sharedlymphocyte activation factor (LAF) activity, and a common major cellularsource, activated macrophages. As information has accumulated fromstudies using purified natural and recombinant IL-1 molecules, it hasbecome clear that IL-1α and IL-1β each mediate most, if not all, of thewide range of activities previously ascribed to IL-1.

TNF-α plays a critical role in the development of acute pulmonaryfailure and injury. When released into the lung, TNF-α has devastatingeffects, causing rapid and diffuse tissue injury. This is presumably adirect result of its known effects on endothelial cells andgranulocytes, as well as its induction of other mediators such as 1IL-1, prostaglandins, and platelet-activating factor.

While one series of embodiments of the present invention employ, as afirst capture reagent, cytokine binding antibodies bound to detectionsurface, the present invention also contemplates the binding of solubleproducts directly to the surface without the use of capture reagents. Instill another series of embodiments, the present invention contemplatesnon-antibody ligands, such as cytokine receptors and lectins (e.g.concanavalin A or lectins), as the capture ligand. With respect tocytokine receptors, the existence of IL-1 plasma membrane receptorswhich bind both IL-1α and IL-1B is now well-established. IL-1 receptorshave now been cloned and expressed in high yield. See S. K. Dower, U.S.Pat. No. 4,968,607, hereby incorporated by reference. Similarly, tumornecrosis factor-α (TNF-α) and TNF-β and their receptors have beenisolated, and DNA sequences encoding these secretory proteins aredescribed. See C. A. Smith et al., European Patent Application No.90309875.4 (Publication No. 0418014A1), hereby incorporated byreference. See also U.S. patent application Ser. Nos. 405,370, 421,417and 523,635, hereby incorporated by reference.

The present invention also contemplates the use of detection substratesto detect and/or screen synthetic cytokine analogues and inhibitors.Cytokine analogues are those compounds that act in an analogous manneras the known cytokine. An example of such an analogue is described inEuropean Patent Application No. 343684, hereby incorporated byreference. See also U.S. patent application Ser. Nos. 266,531, 199,915,238,713, 248,521, and 238,171, hereby incorporated by reference. In thatcase, the analogue is a polypeptide inhibitor of interleukin-1.

Finally, the present invention can be used with success to measure avariety of cellular products other than cytokines. In one embodiment,the present invention contemplates detecting other products of T cellssuch as perforins and granzymes. Perforin is a lytic protein andgranzymes are a family of natural serine proteases; these products arestored in secretory granules until released from the cell. See e.g. G.Berke, “The CTL's Kiss of Death” Cell 81:9-12 (1995).

The present invention also contemplates measuring products from cellsother than T cells. For example, the device and method of the presentinvention can be employed to detect the expression and secretion ofrecombinant products from host cells. Further, the devices and methodsof this invention can be used to detect non-secreted materialsassociated with cell membranes. As described above, cells can be grownin conditions favoring their adherence to a membrane having a capturereagent specific for the analyte to be detected. An analyte can be boundto a cell membrane, such as an integral membrane protein, and after asuitable time in culture, the cell can be removed from the membraneusing lytic methods. Lytic methods include use of detergents, such asTWEEN 80 TM, lipid solvents such as acetone or an alcohol, oralternatively, the cells can be ruptured by hypoosmotic shock, forexample using distilled water. Once the cell contents are washed away,residual, undesired membrane components can be removed with the use ofsolvents or detergents followed by washing. Thus, the dominant materialsleft adhered to the membrane are those bound to a specific capturereagent. Analysis of those materials can be accomplished using methodsdescribed herein.

The methods described herein are also suitable for measuring moleculesreleased by dying cells. When T cells kill a target cell, the contentsof the killed cell spill and can be captured around the dieing cell. Inthis way, the number of cells killed can be exactly enumerated.

The present invention also includes devices and methods for detectingalloimmune responses, and in particular, as a measurement of potentialallograft rejection in the field of human organ transplantation.

In this regard, it should be stressed that up to 30-40% of transplantedorgans are lost over 5 years, mostly due to chronic rejection.Importantly, present HLA matching procedures are based on definingserological or structural differences between HLA alleles, but do notreveal any functional information regarding potential alloreactivity.Detectable single amino acid differences between HLA alleles may, or maynot, be clinically important. There is thus a clear need for improvedtechniques that can provide predictive functional information above andbeyond the present typing 1 technologies.

The present state of clinical organ transplantation requires the use ofimmunosuppressive agents to prevent acute transplant rejection. Theexact pharmacological regimen used for immunosuppression variessignificantly from one transplant center to the next, but generallyincludes cyclosporine A, corticosteroids and azathioprine, in somecombination. Serum or whole blood cyclosporine levels are used primarilyto prevent toxicity and not to evaluate the level of immunosuppression;it is well established that patients with therapeutic cyclosporinelevels can develop acute rejection episodes. Additionally, cyclosporinelevels do not provide any information about the relativeimmunosuppressive effects of the other agents used in conjunction withthe cyclosporine A. There is presently no method for assessing theimmunosuppressive effects of corticosteroids or azathioprine (except toevaluate for signs of clinical toxicity), and medication doses are oftenadjusted empirically. Thus, as is the case with structural-based assaysfor tissue typing, there are presently no methods available tofunctionally assess the quality or quantity of the alloimmune responsein transplant recipients.

Embodiments of the present invention can fill this need. It iscontemplated that detection particle based assays of the presentinvention will measure existing and potential human alloimmune responsesby accurately detecting and quantifying allospecific cytokine profiles.The assay of the present invention has a number of advantages that makeit ideal for the monitoring of T cell activity in vivo. Detection of thecytokine occurs directly at its source, i.e. the secreting cell, beforeit is diluted, degraded, or absorbed to receptors on a bystander cell,thus making it extremely sensitive. Indeed, the assay can detect singleantigen-reactive cells. Secondly, the detection particle based assaymeasures clonal sizes, defining the frequencies of antigen-reactive, oralloreactive T cells in the bulk population. Since the 24 hour durationof the assay is too short for T cell proliferation to occur in vitro,the frequency of detected T cells is reflective of the in vivo 1frequency. Thirdly, the assay identifies the cytokine profile of eachresponding cell. This approach is additionally superior to previouslypublished, time consuming, isolation and characterization of in vitrocultured T cell clones, in that the short incubation time of themicrosphere based assay circumvents in vitro artifacts.

The assay of the present invention is very sensitive. This improvedsensitivity is central to monitoring alloreactivity in a potentiallyinfrequent responding population: immuno-suppressed transplantrecipients. The sensitivity is also central to detecting responsesbetween related and unrelated individuals. By “related” individuals(such as humans) it is meant that such individuals have a commonancestry (e.g. children of the same parents, cousins, etc.). By“unrelated” individuals it is meant that such individuals have no knownancestral relationship.

Embodiments of this invention can also be used in analyses ofinfections, vaccinations for infections, cancer, and autoimmune disease.

In still further embodiments, kits embodying detection reagents,detection particles and other materials needed to carry out assays ofthis invention are provided. In general, a kit comprises: (1) one ormore detection particles, (2) one or more detection reagents, (3) asolution for preparing a mixture of detection particles and detectionreagents, and (4) instructions for use. In other embodiments, adetection substrate can also be supplied.

In particular embodiments, a kit may contain (1) one or more detectionparticles with streptavidin bound thereto, (2) one or more detectionreagents with biotin bound thereto, (3) a reaction vial, (4) a solutionfor reacting the detection particles with the detection reagents, and(4) instructions for use. In alternative embodiments, a kit mayadditionally contain one or more agarose particles with straptavidinattached thereto, wherein the agarose particle has sufficiently greatermass to as to be capable of being sedimented under conditions such ascentrifugation. Such kits may be prepared with all of the necessaryinstructions for preparing the materials to be used in an assay andinstructions for carrying out the assay procedure.

EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); M (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters);ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters);μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); BD (BectonDickenson, Bedford, Mass.); ENDO (Endogen, Cambridge, Mass.); PHRM(Pharmingen, San Diego Calif.).

Example 1 Use of Detection Surfaces in Detection Particle Based Assay

In this example, a detection surface is in a microtiter plate to detectcellular products. First, the detection surface is coated withanti-interferon gamma (IFN gamma antibody 1) as the capture reagent.After several hours of incubation (2 hrs to overnight), during which thecapture reagent(s) bind(s) to the membrane, excess reagent is washedaway. The unsaturated surfaces of the membrane are then blocked withirrelevant protein (bovine serum albumin or gelatin) to preventsubsequent nonspecific binding of proteins. Following the blocking step,the plates are washed to remove non-plate bound, excess blockingreagent.

At this point the detection substrate is properly prepared to testcells. In this case, human peripheral blood mononuclear lymphocytes(PBL) are added at a concentration of about 5×10⁵ per well. Specificantigen (e.g. HIV antigen) is added to the experimental wells, controlwells contain no antigen or irrelevant antigen (e.g. myohemerythrin, aprotein that humans have not encountered). Among all cells plated (about5×10⁵ cells) only the antigen specific T cells will be stimulated by theantigen to release secretory products (e.g. IFN-gamma). In the controlwells, in the absence of the antigen, antigen specific cells are notstimulated and do not release secretory products. During a 4-48 hr cellculture period (dependent on the product to be detected) the antigenspecific cells secrete their products(s) being captured by the capturereagents around the secreting cell.

Following the culture period, the cells are washed away, leaving theirsecretory product retained on the membrane. A detection reagent bound toa detection particle is added to bind to the plate-bound secretoryproduct (the detection and capture antibodies specific for secretoryproducts, e.g., IFNγ have to recognize different parts of the moleculesuch as not to interfere with each other's binding). The detectionparticle is either directly labeled with a fluorochrome (e.g., FITC, PEor texas red) or has a characteristic spectral feature. (e.g., differentspectral features for different cellular products). Directly labeleddetection antibodies associated with detection particles areparticularly useful. Alternatively, an antibody is added that isspecific for the analyte and a secondary antibody is attached to thedetection particle. Coupling of detection antibody to the detectionparticle by biotin-streptavidin can also be used.

The present invention contemplates a variety of means for visualizingthe detection particles using a detection reagent such as: a) immunefluorescence if the secondary (or tertiary) reagent was a fluorochromeor b) a characteristic spectral feature (e.g., different “colors” fordifferent analytes) visualized by light microscopy. Each “spot” ofcolored substrate corresponds to one cell producing or secreting theproduct. The difference in number of spots between antigen stimulatedwells and the number of spots in the negative control wells (with mediumalone or irrelevant antigen) establishes the “signal” to be analyzed(usually there is no spot formation in the negative control). Sizes ofthe spots correspond to the quantity of product secreted, the number ofspots establishes the frequency of antigen specific cells in the cellpopulation tested (i.e. cells that were induced by the antigen tosecrete the product among all cells plated, e.g., 20 IFN-gamma spots inHIV antigen challenged PBL represent a frequency of 40/million, if 5×10⁵cells were plated).

Example 2 Peptide Screening

In this example, microwells are employed to screen and identify theantigenic determinants (i.e. MHC-bound peptides of the antigen generatedby intracellular processing) that T cells recognize, using peptides.Usually, in studies that involved peptides, a few randomly chosensequences of the antigen are tested as peptides. However, evenoverlapping sequences that walk down the molecule in steps of 5 to 10amino acids do not necessarily detect all determinants. By contrast, inthis example overlapping sets of peptides are employed that walk themolecule amino acid by amino acid. This peptide scan includes everypossible determinant on an antigen and provides an exact mapping of theT cell repertoire.

While an assay system has been employed to test for antigen specific CD4cells [G. Gammon et al., “T Cell Determinant Structure: Cores andDeterminant Envelopes in Three Mouse Major Histocompatibility ComplexHaplotypes” J. Exp. Med. 173: 609-617 (1991)], the present inventionallows for testing of CD8 cells. CD8 determinant mapping for HIVidentifies possible peptides for vaccines. Determinant mapping forautoantigens in autoimmune disease has prognostic and possibletherapeutic consequences.

For determinant mapping, Myelin Basic Protein (“MBP”) peptides that walkthe MBP molecule amino acids by amino acid are used. These peptidesproduce clear data in SHIVERER mice. The peptides can be frozen withoutlosing bioactivity at 14 μM concentration in HL-1 medium, which is 2×the optimal concentration for the bioassay. Thus, the large number ofpeptides involved do not have to be diluted and plated for eachexperiment, but can be freshly thawed from storage. The entire peptideseries is diluted, pipetted and frozen series in advance and, on the dayof experiment, the required number of plates are prepared.

The number of cells available from patients can be limiting; one millioncells can be obtained from one milliliter of blood and usually fiftymilliliters of blood is available per patient (i.e., fifty million cellsmay be the total cells available for the assay). Because 2×10⁵ cells perwell provides clear results with the plate of the present invention,this is sufficient for testing 250 peptides, which constitutes anaverage size protein. However, by miniaturizing the plates (e.g. to 50microliter wells), one can test for 1000 peptides, which can cover eventhe larger proteins. The entire peptide series can be tested on anindividual. The plating of the cells can be performed with 12 channelpipettors into the freshly prepared plates, reducing the timeinvolvement on the first day of the assay to a couple of hours.

In this example, PBL cells were plated at 2×10⁵ cells per well into 96well microtiter plates containing the hydrophobic membrane pre-coatedwith an antibody as capture reagent. The cells are incubated in thewells with antigen for 24 h at 37° C., 8% CO₂. During this cultureperiod, T cells with specificity for a given peptide are activated andstart secreting cytokines and other cellular products, which arecaptured by the appropriate antibody on the plate around producingcells. Thus, secreting cells (antigen specific memory cells) aresurrounded by a “spot” of the cytokine.

After an additional culture (24 hours), the cells are then washed awayand the bound cytokine is detected by a biotinylated second antibodythat is specific for the same cytokine but recognizes a differentdeterminant on it. The plate bound second antibody is attached to amicrosphere with a characteristic marker thereon.

The numbers of spots in antigen containing wells vs. wells containingmedium alone or irrelevant antigens establish the number of antigenspecific cells in the culture. Since 2×10⁵ cells are plated to eachwell, the absolute frequency represents the frequency of antigenreactive T cells in 2×10⁵ T cells of the given specificity within thecell pool tested.

Example 3 Two Color Detection Particle Based Assay

In this example, a capture surface is prepared as in Example 1, exceptthat a second capture reagent and a second detection reagent/microsphereis used as well. To prepare the surface for a two-color assay, two (2)capture reagents (anti-IFNγ and anti-IL-5 antibody) are usedsimultaneously (for additional capabilities, still additional capturereagents can be employed for a “multicolor” assay).

Cells are again plated (about 5×10⁵ cells) and only the antigen specificT cells are stimulated by the antigen to release secretory products.However, in this case, the surface is capable of detecting both IFNγ andIL-5. After the culture period and washing described in Example 1, asecond detection reagent attached to a second detection particle isemployed together with the first detection reagent attached to a firstdetection particle. In this case, detection particles labeled withanti-IL-5 are added (alternatively, fluorochromes emitting differentcolors when excited by UV light permit two or multicolor assays e.g.,FITC:green, PE:red). Spots originating from cells that secreted one ofthe products only, will appear as a single color (e.g., red or blue),while cells secreting both will have the both colors represented bydifferent detection particles.

Example 4 Mixed Lymphocyte Culture

In this example, the detection particle based assay of the presentinvention is used as a functional measurement of alloimmune responses.Alloresponses are measured in naive responder mouse splenocytes uponstimulation with irradiated allogeneic stimulator splenocytes. A Balb/c(H-2^(d)) anti-SJL (H-2^(S)) mixed lymphocyte response (MLR) is set upby mixing 8×10⁵ responder splenocytes (H-2^(d)) with 4×10⁵ irradiatedstimulator splenocytes (H-2^(s)) in 200 micro Liter of HL-1 media (serumfree), in 96 well plates that were pre-coated with antibodies to IL-2,IL-5 or IFNγ as capture reagents.

A strong proliferative response is detected and the secretion of Th1(IFN-gamma), but not Th2 (IL-5) cytokines, is measured in culturesupernatants. In addition, there is CTL killing of SJL target cells.

The detection particle based assay can be evaluated visually using amicroscope or other optical device. The Balb/c (H-2^(d)) anti-SJL(H-2^(s)) response is primarily of a Th1 phenotype, with strong IL-2 andIFN-gamma 10 but essentially no IL-5 production. Control wells withstimulators alone, responders alone, and H-2^(d) stimulators mixed withsyngeneic, irradiated H-2^(d) responders all reveal no spots for anycytokine. Similar assays are performed in multiple strain combinationswith equivalent results, showing that the quality of the response is notstrain specific. Quantitative analysis (spots per 10⁶ splenocytes) foreach cytokine reveals 180-250 spots per million responder splenocytesplated for both IFNγ and IL-2, while <5 per million IL-5 spots aredetected. Notably, these responses are 5-10-fold greater than theantigen-specific responses (which are typically 15-30 spots per millioncells) from the same mouse strain.

In order to verify that the assay is quantitatively reliable, a similardetection particle based analysis is performed on serially dilutedsamples, and we find that the number of responding cells falls linearlyin relation to the serial dilution. Thus, the detection particle basedassay yields reproducible quantitative and qualitative informationregarding the alloimmune response.

Example 5 Human Transplant Recipients

Given the ability of embodiments of the present invention to detectantigen-specific responses, the methods are also suitable for the studyof human alloresponses in the transplant setting, including but notlimited to the testing of immunosuppressed transplant recipients. Thepresent invention contemplates the use of the microsphere based assay asa) a functional predictor of allograft survival in cadaver and livingrelated renal transplant recipients, and b) a tool for monitoring thelevel of immunosuppression in renal allograft recipients.

96 well plates are coated with capture reagent for either IL-2, IL-4,IL-5, IL-10, or IFN in PBS. Peripheral blood lymphocytes (PBLs) areprepared from normal human volunteers, mixed in variousstimulator/responder ratios in 2 mls of complete RPMI in a 24 wellplate. After a 12-72 hour activation (IL-2 and IFN can best be detectedat 12-24 hours, while IL-4, IL-5, and IL-10 require 36-72 hours, andmore in other embodiments, 48 hours), the cultures are harvested,washed, and added to the precoated plates. After another 24 hours thecells are washed from the plates and second detection antibodiesattached to microspheres are added to the wells. The plates are washedafter an overnight incubation, and after a final wash, cytokine-specificspots are analyzed by the image analysis computer program as describedabove.

While it is not intended that the assays of the present invention belimited by the use of particular antibodies, work with human PBLs hasresulted in the determination that the antibody combinations set forthin Table 2 can effectively detect human cytokines in recall responses tocommon antigens.

TABLE 2 Coating antibody Detection antibody Cytokine (Capture Reagent)(Detection Reagent) IL-2 BG-5 (Serotec) Rabbit polyclonal (BD) IL-48D4-8 (PHRM) MP4-25D2 (PHRM) IL-5 JES1-39D10 (PHRM) JES1-5A10 (PHRM)IL-10 JES3-9D7 (PHRM) JES3-12G8 (PHRM) IFN MA700 (ENDO) MA701 (ENDO)

Blood samples are obtained from normal human volunteers in heparinizedgreen top tubes, and rocked gently at room temperature until ready foruse. PBLs are prepared by standard Ficoll Hypaque centrifugation andcounted. By these methods, one can obtain between 1×10⁶ and 3×10⁶ PBLsfrom each milliliter of blood. All experiments are performed usingcomplete RPMI media.

In some instances, T cell enrichment is desirable. T cells can beenriched from PBLs using human T cell isolation columns (R and DSystems), as per manufacturer's recommendations. In other instances,depletion of T cell subtypes is desired. Depletion of T cell subtypescan be performed by standard methods, using OKT3 (anti-CD3), OKT4(anti-CD4), or OKT8 (anti-CD8), and rabbit complement (Cedarlane,Hornsby Ont). Aliquots of the resultant cells can be washed and stainedwith FITC conjugated antibodies for phenotyping by FACS. The antibodiesare isolated from hydriboma supernatants grown in our laboratory bystandard methods.

The alloimmune responses in normal individuals can produce primarily Th1type cytokines, i.e., IL-2 and IFN, with little IL-4, IL-5 or IL-10. Itis expected that detected alloreactive responder frequencies in normalindividuals will fall in the range of 0.1-10% of the responding T cells.The results for immunosuppressed individuals may vary, but it isexpected that the cytokine production will decrease in strength and theclonal size of the response will also be relatively decreased. Withsufficient numbers of normal individuals for comparison, animmunosuppressed individual who shows very little relative decrease inresponse may show signs of allograft rejection.

Example 6 Two Color Assay for Human IL2 and Human Interferon γ

On day 1 plates (MIP45, Millipore) were coated simultaneously with twoprimary capture monoclonal antibodies (mAbs), one raised against (1)h-IFN-γ. Endogen MA700A) at a concentration of 4 μg/ml and anotherraised against h-IL-2 (R&D Systems 5334.21) at a concentration of 3μg/ml. 200 μl of coating mAbs mixture was added per well. Plates werecoated overnight at 4° C.

On day 2 the plates were washed 3 times with PBS (200 μl/well), andblocked with 1% BSA in PBS for 2 h at RT (200 μl/well). PPD antigen wasadded to the well at concentration 100 μg/ml (100 μl/well). 300.000human unseparated PBMCs were added to the same wells (100 μl/well).Plates were incubated for 24 h at 37° C., humidified, in the presence of7% CO₂.

On day 3 cells and unbound materials were removed from the wells bywashing 3 times with PBS and 3 times with PBS-Tween (0.05%). Then platewas incubated overnight at 4° C. with 100 μl per well of detectionparticles directly labeled with secondary detection reagents beingantibodies. Red dots (655 nm) were labeled with anti h-IFN-γEndogenM701) and used at a concentration 5.0×10⁻⁹ M. Green dots (525 nm) werelabeled with h-IL-2 mAbs (Endogen BG5) and used at a concentration of1.0×10⁻⁸ M.

On day 4 unbound detection particles were washed out 4 times withPBS-Tween (0.05%) and 3 times with PBS. Digital images were taken usinga Fluorescent microscope (Optem) with a long-path emission filter(Chroma 32013).

FIG. 3 depicts results of such a study. A photomicrograph of a welltreated as above shows red dots (R) that indicate the presence ofsecreted h-IFN-γ. Green dots (G) indicate the presence of secretedh-IL-2.

Example 7 Use of Nano-Particles Labeled with Streptavidin for MulticolorAssay of Cell-Secreted Products

In other embodiments, methods can be used to substitute particlesdirectly labeled with detection secondary antibodies. It is analogous tothe indirect method described here, with the difference that interactionof the streptavidin-labeled particles with biotinylated secondaryantibodies takes place prior to their interaction with thecytokine-primary coating antibody complex on the surface.

The method is based on the fact that interaction of streptavidin withits natural ligand, biotin, is practically irreversible; the equilibriumbinding constant for this interaction exceeds the magnitude of 10¹¹ M⁻¹.

Particles of each individual type (color) covalently conjugated withstreptavidin or its analog, avidin, are premixed with the biotin-labeledsecondary detection antibody (reagent) specific to each secretedproduct. After the reaction is completed, the excess of unboundbiotinylated antibodies is removed by the addition of agarose beadscoated with streptavidin. Agarose beads with the bound excessivebiotinylated antibodies are then removed by centrifugation. Particles ofdifferent color, each labeled with single type of the secondarydetection antibody (reagent) through streptavidin-biotin interaction,are incubated with the surface-bound secreted product similar to the waydirectly conjugated particles are used. Several particle types can beused either simultaneously or in a number of consecutive steps.

Example 8 Preparation of Purified Detection Particles Labeled withStreptavidin and Biotinylated Detection Antibodies

In other embodiments, this invention includes new methods for producingdetection particles having detection reagents thereon. An embodiment ofthis method is illustrated in FIG. 4. A nanoparticle 404 labeled withstreptavidin 405 is mixed with biotinylated detection reagent 408comprising biotin 409 coupled to a detection reagent 410. These areincubated together for sufficient time to permit biotin 409 ofbiotinylated detection reagent 408 to bind streptavidin 405 on thenanoparticle 404 thereby forming biotinylated streptavidin detectionparticle complex 411. Some free biotinylated detection reagent 408 isdepicted free in solution. Then, an agarose particle 414 havingstreptavidin 405 thereon is added to the mixture and incubated for afurther period of time. The resulting solution contains complexes 411and agaroselbiotinylated detection reagent complexes 418. Little or nofree biotinylated detection reagent 408 is present in the solution.Next, centrifugation step 420 results in sedimentation ofagarose/biotinylated detection reagent complexes at 424, leavingbiotinylated streptavidin detection particle complexes 411 free insolution. Subsequently, complexes 411 can be used in detection particlebased assays.

From the above, it should be clear that the present invention providesimproved devices and methods to measure secreted cell products, and inparticular, secreted T cell cytokines. The devices and methods of thepresent invention provide a greater capability to detect cytokines fromindividual cells in a mixture of heterogeneous cells. The methods alsopermit more accurate, rapid, reproducible assays that can be used todetect multiple secreted cellular products in the same assay by usingmultiple detection particles each having a characteristic spectralfeature. It can be appreciated that the above descriptions are providedby way of example only, and are not intended to limit the scope of theinvention. Persons of ordinary skill can take the teachings of thisapplication and produce modifications and variations that are within thescope of this invention. All of those modifications and variations areconsidered to be part of this invention.

1. A method of detecting a secreted cell product, comprising: a)providing a reaction chamber comprising: cell culture and detectionsubstrate upon which a cell that produces and secretes the cell productcan adhere and a complex formed by the cell product with a detectionparticle is visualized as a spot on said substrate; said detectionsubstrate having capture reagent thereon that binds to said cell productto form a product-capture reagent complex; b) adding a sample of cellsto said reaction chamber and permitting at least one of said cells toadhere to said substrate; c) stimulating said cells to produce saidproduct, wherein said product binds to said capture reagent to form aproduct-capture reagent complex at a location near said cell on saidsubstrate and then washing away said cells to expose said location; d)adding a detection reagent to said reaction chamber, wherein saiddetection reagent binds to said product producing a detectionreagent-cell product-capture reagent complex (DRC); e) adding adetection particle to said chamber, wherein said detection particlebinds to said detection reagent via streptavidin/biotin bindingproducing a detection particle-detection reagent-cell product-capturereagent complex (DRCC); and f) visualizing said detection particle onsaid DRCC as a spot on said substrate.
 2. The method of claim 1, whereinsaid capture reagent is a product-specific ligand covalently attached tosaid substrate.
 3. The method of claim 2, wherein said product-specificligand is an antibody or a product-specific receptor.
 4. The method ofclaim 2, wherein said detection substrate comprises a microwell.
 5. Themethod of claim 1, wherein said capture reagent is a surface, and saidproduct becomes associated with said surface by way of physicalinteraction.
 6. The method of claim 5, wherein said physical interactionis hydrophobic interaction.
 7. The method of claim 1, wherein saidproduct is an interferon.
 8. The method of claim 7, wherein saidinterferon is interferon gamma.
 9. The method of claim 1, wherein saidproduct is an interleukin.
 10. The method of claim 9, wherein saidinterleukin is IL-5.
 11. The method of claim 1, wherein said cell is ahuman cell.
 12. The method of claim 1, wherein said detection particleis a nanoparticle having a lower limit of size of about 90 Å and anupper limit of about 500 Å.
 13. The method of claim 1, wherein saiddetection particle is a microparticle having a lower limit of size ofabout 0.05 μm; and an upper limit of size of about 5 μm.
 14. The methodof claim 1, wherein said particle is made of a material selected fromthe group consisting of organic polymers, inorganic semiconductors andheavy metals.
 15. The method of claim 14, wherein said organic polymeris selected from the group consisting of polystyrene, polyacrylamide,polyacrolein and polycarbonate.
 16. The method of claim 14 wherein saidinorganic semiconductor is selected from the group consisting of silicondioxide-containing semiconductors and cadmium selenide (CdSe).
 17. Themethod of claim 14, wherein said heavy metal is selected from the groupconsisting of gold, silver, platinum, palladium, and lead.
 18. Themethod of claim 1, wherein said detection particle exhibits a spectralproperty selected from the group consisting of absorption, fluorescenceand phosphoresence.
 19. The method of claim 18, wherein said detectionparticle exhibits a spectral feature having a lower limit of wavelengthin the ultraviolet portion of the spectrum and an upper limit in theinfrared portion of the spectrum.
 20. A method for determiningalloreactivity, comprising a) providing a reaction chamber comprising:cell culture and detection substrate upon which a cell that produces andsecretes the cell product can adhere and a complex formed by the cellproduct with a detection particle is visualized as a spot on saidsubstrate; said detection substrate having a capture reagent thereonthat binds to said cell product to form a product-capture reagentcomplex; b) adding a first cell to said reaction chamber; c) adding asecond cell to said reaction chamber, where at least one of said firstcell and said second cell adheres to said substrate, wherein one of saidcells stimulates said other of said cells to produce a cellular product,wherein said cellular product binds to said capture reagent to form aproduct-capture reagent complex at a location near at least one of saidcells; d) washing away said cells to expose said location; e) adding adetection reagent to said reaction chamber, wherein said detectionreagent binds to said product producing a detection reagent cellproduct-capture reagent complex (DRC); f) adding said detection particleto said chamber, wherein said detection particle binds to said detectionreagent via streptavidin/biotin binding producing a detectionparticle-detection reagent-cell product-capture reagent complex (DRCC);and g) visualizing said detection particle on said DRCC as a spot onsaid substrate, wherein presence of a cellular product which resultsfrom the one cell stimulating another cell provides indication ofalloreactivity.
 21. The method of claim 20, wherein said first cell andsaid second cell are derived from related human beings.
 22. The methodof claim 20, wherein said first cell and said second cell are derivedfrom unrelated human beings.
 23. A method of detecting a plurality ofsecreted cell products, comprising: a) providing a reaction chambercomprising: cell culture and detection substrate upon which cells thatproduce and secrete the cell products can adhere and complexes formed bythe cell products with distinct detection particles are visualized asspots on said substrate; said detection substrate having a plurality ofcapture reagents thereon, each of said reagents binds to at least one ofsaid plurality of secreted cell products; b) adding a sample of cells tosaid reaction chamber, where at least one of said cells adheres to saidsubstrate; c) stimulating said cells to produce said plurality of cellproducts, wherein said products bind to at least two of said capturereagents to form product-capture reagent complexes at a location near atleast one of said cells and then washing away said cells to expose saidlocation; d) adding a plurality of distinct detection reagents to saidreaction chamber, wherein each of said detection reagents binds to onetype of said cell products forming at least a first complex and a secondcomplex of said cell products and said detection reagents, producing aplurality of detection reagent-cell product-capture reagent complexes(DRC); e) adding a plurality of types of detection particle to saidchamber, wherein each of said types of detection particle binds viastreptavidin/biotin binding to only one of said detection reagentsproducing a plurality of detection particle-detection reagent-cellproduct-capture reagent complexes (DRCC), and wherein each distinct typeof detection particle has a different spectrographic feature associatedtherewith sufficient to permit differential detection of each of saidplurality of complexes; and f) visualizing said plurality of detectionparticles on said complexes as one or more spots on said substrate. 24.The method of claim 23, wherein each of said plurality of capturereagents comprise antibodies specific for one of said plurality of cellproducts.
 25. The method of claim 23, wherein said capture reagentcomprises a surface to which said cell products become associatedthrough physical interaction.
 26. The method of claim 25, wherein saidphysical interaction is hydrophobic interaction.
 27. A kit for detectingthe presence of a cell product comprising: a cell culture reactionchamber comprising: a detection substrate upon which a cell thatproduces and secretes the cell product can adhere and a complex formedby the cell product with a detection particle is visualized as a spot onsaid substrate; said detection substrate having a capture reagentthereon that binds to said cell product forming a product-capturereagent complex; a detection reagent that binds to said cell product orsaid product-capture complex; a detection particle having acharacteristic spectrographic feature adapted to bind to said detectionreagent via streptavidin/biotin binding to thereby form a detectionparticle-detection reagent complex; a solution for mixing said detectionparticle and a solution for mixing said detection reagent; andinstructions for use, said instructions including the following steps:a) adding a sample of first cells to said reaction chamber, where atleast one cell adheres to said substrate; b) stimulating said cell toproduce said product, wherein said product binds to said capture reagentto form a product-capture reagent complex at a location near said cellon said substrate; c) washing away said cell to expose said location; d)adding said detection reagent to said reaction chamber, wherein saiddetection reagent binds to said product, producing a detectionreagent-cell product-capture reagent complex (DRC); e) adding saiddetection particle to said chamber, wherein said detection particlebinds to said DRC, producing a detection particle-detection reagent-cellproduct-capture reagent complex (DRCC); and f) visualizing saiddetection particle on said DRCC as a spot on said substrate.
 28. The kitof claim 27, further comprising at least one solution suitable forgrowing cells.
 29. The kit of claim 27, wherein said capture reagent isa product-specific ligand covalently attached to said substrate.
 30. Thekit of claim 29, wherein said product-specific ligand is an antibody ora product-specific receptor.
 31. The kit of claim 29, wherein saiddetection substrate comprises a microwell.
 32. The kit of claim 27,wherein said capture reagent is a surface, and said product is adsorbedsaid surface by way of physical interaction.
 33. The kit of claim 32,wherein said physical interaction is hydrophobic interaction.
 34. Thekit of claim 27, wherein said product is an interferon.
 35. The kit ofclaim 34, wherein said interferon is interferon gamma.
 36. The kit ofclaim 27, wherein said product is an interleukin.
 37. The kit of claim36, wherein said interleukin is IL-5.
 38. The kit of claim 27, whereinsaid cell is a human cell.
 39. The kit of claim 27, said instructionsfurther comprising, after step a): adding a second cell to said reactionchamber, wherein one of said first or second_cells stimulates another ofsaid cells to produce a cellular product indicative of alloreactivity,wherein said cellular product binds to said capture reagent.
 40. The kitof claim 39 having instructions for use, wherein said first cell andsaid second cell are derived from related human beings.
 41. The kit ofclaim 39 having instructions for use, wherein said first cell and saidsecond cell are derived from unrelated human beings.
 42. The kit ofclaim 27, wherein said detection particle is a nanoparticle having: alower limit of size of about 90 Å; and an upper limit of about 500 Å.43. The kit of claim 27, wherein said detection particle is amicroparticle having: a lower limit of size of about 0.05 μm; and anupper limit of size of about 5 μm.
 44. The kit of claim 27, wherein saidparticle is made of a material selected from the group consisting oforganic polymers, inorganic semiconductors and heavy metals.
 45. The kitof claim 44, wherein said organic polymer is selected from the groupconsisting of polystyrene, polyacrylamide, polyacrolein andpolycarbonate.
 46. The kit of claim 44, wherein said inorganicsemiconductor is selected from the group consisting of silicondioxide-containing semiconductors and cadmium selenide (CdSe).
 47. Thekit of claim 44, wherein said heavy metal is selected from the groupconsisting of gold, silver, platinum, palladium, and lead.
 48. The kitof claim 27, wherein said detection particle exhibits a spectralproperty selected from the group consisting of absorption, fluorescenceand phosphorescence.
 49. The kit of claim 48, wherein said detectionparticle exhibits a spectral feature having a lower limit of wavelengthin the ultraviolet portion of the spectrum and an upper limit in theinfrared portion of the spectrum.
 50. A kit for detecting the presenceof a plurality of secreted cell products, comprising: a reaction chambercomprising: a cell culture substrate upon which cells that produce andsecrete the cell products can adhere and complexes formed by the cellproducts with distinct detection particles ate visualized as spots onsaid substrate; said substrate having a plurality of capture reagentsthereon, that each bind to at least one of said plurality of secretedcell products to form a product-capture reagent complex; a plurality ofdetection reagents, that are each adapted to bind to one type of saidcell products to thereby from a plurality of detectionparticle-detection reagent complexes; a plurality of types of distinctdetection particles, wherein each of said types of detection particlesbinds via streptavidin/biotin binding to only one of said detectionreagents to thereby form a plurality of detection particle-detectionreagent complexes, and wherein each type of distinct type of detectionparticles has a spectrographic feature associated therewith sufficientto permit differential detection of each of said plurality of saidcomplexes; a solution for mixing said plurality of detection reagentsand a solution for mixing said plurality of detection particles; andinstructions for use, said instructions including the following steps:a) adding a sample of cells to said reaction chamber, at least one ofsaid cells adhering to a location on said substrate; b) stimulating atleast one of said cells to produce said plurality of cell products,wherein each of said products binds to at least one of said capturereagents to form product-capture reagent complexes near the location ofsaid cell; c) washing away said cell to expose said location; d) addingsaid plurality of detection reagents to said reaction chamber, whereineach of said detection reagents binds to one type of said cell products,forming a at least a first complex and a second complex of said productsand said detection reagents, producing a plurality of detectionreagent-cell product-capture reagent complexes (DRC); e) adding saidplurality of types of detection particles to said chamber, wherein eachof said types of detection particles binds to only one of said DRCcomplexes producing a plurality of detection particle-detectionreagent-cell product-capture reagent complexes (DRCC); and f)visualizing said distinct detection particles on said complexes as oneor more spots on said substrate.
 51. The kit of claim 50, wherein eachof said plurality of capture reagents comprise antibodies specific forone of said plurality of cell products.
 52. The kit of claim 50, whereinsaid capture reagents comprise a surface onto which said cell productsbecome adsorbed through physical interaction.
 53. The kit of claim 52,wherein said physical interaction is hydrophobic interaction.